It is of great agricultural and economic interest to provide new plants which display an improvement in particular characteristics. Through proper breeding techniques, these characteristics can be introduced into new or existing genotypes of plant species which can then be marketed directly or used to produce superior hybrid plants.
The development of a hybrid variety conventionally involves three steps: (1) the selection of superior plants from various germplasm pools; (2) the selfing of the superior plants for several generations to produce a series of inbred lines, which although different from each other, breed true and are highly uniform; and (3) the crossing of selected inbred lines with unrelated inbred lines to produce the hybrid progeny (F.sub.1). An important consequence of the homozygosity and homogeneity of the inbred lines is that the hybrid between any two inbreds will always be the same. Once the inbreds that give the best hybrid have been identified, the hybrid seed can be reproduced indefinitely as long as the homogeneity of the inbred parent is maintained.
There has been much discussion about the potential utilization of haploids in plant breeding. Since plant breeding is concerned with the development of genotypes to use directly or as parents of productive hybrids, the rapid advance to homozygosity which accompanies the doubling of haploids is an attractive feature. However, attempts at utilizing haploids in plant breeding have been frustrated by the lack of a reliable means of generating sufficient numbers of doubled haploid lines from a broad spectrum of commercially-important germplasm.
Anther culture provides a rapid method of inducing homozygosity in plants which are of interest for the production of breeding lines. Anther culture involves isolating immature anthers from plants and placing them onto a medium which induces the cells within the anther, which would normally be destined to become pollen grains, to begin dividing and form a cell culture from which plants can be regenerated. For a general discussion of anther culture, see J. M. Dunwell, "Anther and Ovary Culture", In S. W. J. Bright and M. G. K. Jones, (eds.), Cereal Tissue and Cell Culture, Martinus Nijhoff Publisher, 1985, Dordrecht, pp. 1-44. This process is known as androgenesis. The resulting cultures are haploid and contain only a single set of chromosomes from the original plants. The plants derived from these cultures are sterile unless chromosome doubling occurs, either spontaneously or by induction, to create doubled haploids which are fully fertile and completely inbred. Therefore, anther culture represents a potentially powerful method of rapidly producing large numbers of inbred lines for commercial evaluation.
Numerous studies on the in vitro culture of gametophytic cells with the aim of producing haploid plants have been reported during the last two decades. A large number of reviews, book chapters and symposia proceedings have been published as well (see generally Chu, "Haploids in Plant Improvement", In I. K. Vasil, W. R. Scowcroft, K. J. Frey (eds.), Plant Improvement and Somatic Cell Genetics, New York: Academic Press, 1982, pp. 129-158; Heberle-Bors, "In Vitro Haploid Formation of Pollen: A Critical Review", Theor. Appl. Genet. 71:361-374, 1985; and Hu and Yang, "Haploids of Higher Plants in Vitro." Berlin, Heidelberg, Springer-Verlag, (1986)).
Anther culture represents a method by which, theoretically, large numbers of haploid individuals can be produced directly from anthers and/or microspores in vitro. (see Keller et al. "Haploids from gametophytic cells --recent developments and future prospects", In C. E. Green, D. A. Somers, W. P. Hackett, D. D. Biesoer (eds.), Plant Tissue and Cell Culture, Alan R Liss, New York, pp 223-241). Haploids can be regenerated from both male and female gametophytic cells through the culture of anthers, microspores, ovaries and ovules. A positive in vitro response will lead to the development of embryos and/or callus from which plants can be regenerated. Early events during in vitro culture have been characterized at the cytological, ultrastructural and biochemical level (Chen et al., "Segmentation Patterns and Mechanisms of Genome Multiplication in Cultured Microspores of Barley", J. Can, Genet. Cytol., 26:475-483 (1984); Raghavan, Protein Synthetic Activity during Normal Pollen Development and During Induced Pollen Embryogenesis in Hyoscyamus niger", J. Can Bot., 1984, 62:2493-2513; Huang, "Ultrastructural Aspects of Pollen Embryogenesis in Hordeum, Triticum and Paeonia", 1986).
Anther culture has been employed to obtain microspore-derived callus, embryos and plants in well over 200 species (Maheshwari et al., "Haploids from Pollen Grains-Retrospect and Prospect", Amer. J. Bot. , 1982, 69:865-879). However, the anther culture responsiveness varies considerably among species. A comparison of the overall responsiveness of anther culturability is made difficult, as the results reported in published studies are given in different bases. For example, anther culturability has been defined by the induction of microspores that begin dividing, the number of embryos and/or callus per anther, the percentage of anthers producing at least one embryo and/or callus, the number of haploid plants regenerated, and the number of dihaploid plants recovered.
The highest yield of responding anthers (anthers forming embryos and/or callus per 100 anthers plated) was found to be 87 percent in wheat (A. M. Wei, "Pollen Callus Culture in Triticumaertivum", Theor. Appl. Genet., 63, 1982, pp. 71-73), 67 percent in rice (S. L. Lin and H. S. Tsay, 1983, J. Agr. Res., China, cited in Dunwell, 1985), 17 percent in maize (Ting et al., "Improved Anther Culture of Maize" (Zea mays L.), Plant Science Lett., 23, 1981, pp. 139-145) and 1 percent in barley (Z. H. Xu and N. Sunderland, "Innoculation Density in the Culture of Barley Anthers", Scient. Sinic., 25, 1982, pp. 961-968). In rye, 43 developing structures per 100 anthers were observed (G. Wenzel et al. , "Increased Induction and Chromosome Doubling of Androgenetic Haploid Rye", Theor. Appl. Genet., 51, 1977, pp. 81-86). Concerning plant regeneration, Petolino and Jones (J. F. Petolino and A. M. Jones, "Anther Culture of Elite Genotypes of Maize", Crop Science, 26, 1986, pp. 1072-1074) describe for maize that from 234 embryoids (from different genotypes) transferred to regeneration medium, 43 developed into plants. Frequencies of calli producing green plant per 100 cultured anthers are in wheat 72 percent (J. W. Ouyang et al., "The Response of Anther Culture to Temperature in Triticum Aestivum", Theor. Appl. Genet., 66, 1983, PP. 101-109), in rice 12 percent (L. J. Chen et al., "Medium Evaluation for Rice Anther Culture", in A. Fujiwara (ed.), "Plant Tissue Culture", pp. 551-551. Jap. Assoc. Plant Tissue Culture Tokyo, 1982) and in barley 10 percent (K. N. Kao, "Plant Formation from Barley Anther Cultures with Ficoll Media", Z. Pflanzenzuchtg., 103, 1981, pp. 437-443).
Although relatively rapid progress has been made in several species, many species of plants, unfortunately, have not shown detectable or significant anther culturability. Production of positive results in maize anther culture has been particularly slow (Nitsch et al., "Production of Haploid Plants Zea mays and Pennisetum through Androgenesis", In E. D. Earle, Y. Demarley (eds.) Variability in Plants Regenerated from Tissue Culture, Prager Publishers, New York, 1982, pp. 69-91). Response frequencies in cultured maize anthers have been very low in all but a few genotypes (see Ku et al. , "Induction Factors and Morpho-cytological Characteristics of Pollen-derived Plants in Maize", (Zea mays L. Proc Symp Plant Tissue Cult., (1978) Science Press, Peking, pp 35-42; Genovesi et al. , "In vitro Production of Haploid Plants of Corn via Anther Culture", Crop Science, 22, 1982, pp. 1137-1144, Dieu and Beckert, 1986; and Petolino et al., "Anther Culture of Elite Genotypes of Maize", Crop Science, 26, 1986, pp. 1072-1074).
Maize genotypes differ with respect to their amenability to anther culture (Petolino et al., 1986, supra) suggesting that genetic factors are important in determining the level haploid production. For anther culture-derived lines to be utilized in commercial maize breeding, commercially-acceptable germplasm will require increased responsiveness to anther culture. Specifically, any attempt to use anther culture in commercial breeding will require a considerable improvement in the overall efficiency of doubled haploid seed recovery. Generally, the major problems in the use of anther culture have been the relatively low response frequencies and the difficulties associated with plant regeneration and chromosome doubling in all but a few genotypes.
As can be seen from the above discussion, anther culture techniques are still rather empirical, and as such it is difficult to draw generalizations from the prior art.
It is thus an object of the invention to provide process for producing germplasm exhibiting enhanced response to anther culture.
It is an object of the invention to provide anther-derived, plants and seed.
Finally, it is an object of the invention to transfer the germplasm providing increased anther culturability from at least one plant in a species to other plants in the species.